Project Summary While new technologies for detecting pathogens are often reported, these typically require small volumes of concentrated and clean samples which can make them impractical to use. The long-term goal is to develop pragmatic, low-cost and easy-to-use assays to identify, separate, concentrate, and detect low concentrations of target bacteria in liquid samples The objective of this application is to use synthetic biology to overcome current obstacles in phage-based detection including sensor performance and host resistance. Two specific aims have been developed towards this objective, 1) Engineer an E. coli-specific phage to produce a cellulose- binding reporter enzyme to enable a ?Lab on a Filter? detection assay, and 2) Engineering bacteriophages to avoid host resistance. By considering a filter to be a reaction surface, a ?Lab on a Filter? concept which can rapidly reduce the time to results and provide low concentration quantification of bacteria in enabled. Bacteriophages (phages) are viruses which infect bacteria, and can be engineered to deliver genes for reporter enzymes to target filtered bacteria during an assay. The enzymes would be overexpressed and released by the bacterial host during the infection. Enzymes fused with a cellulose-binding module would immobilize directly on a cellulose filter in proximity to the lysed bacteria. Enzyme-reactive precipitating dyes can then be used to form colored precipitate in the proximity of the immobilized enzymes. The result is a fully quantitative (0 ? 250 CFU/100 mL) and assay for bacteria which is amenable to both standard and non-laboratory settings and can be provide results after only a few hours. Phages which target and kill specific bacteria exist for almost all known bacterial pathogens. The use of phages for both bacteria detection and for combating multidrug resistant bacterial infections continues to increase. The main hurdle with using phages for this purpose, is the ability of the bacterial host to evolve resistance through random mutations of surface antigens. The ability to genetically engineer phages to avoid host resistance will have a significant and positive impact on phage- based pathogen detection as well as phage therapy to treat multidrug-resistant-bacterial infections. By engineering a phage to have multiple surface recognition receptors (tail fibers), the bacterial host would require several mutations to avoid adsorption of the phages. This can be performed by engineering mixed tail fibers targeting the same pathogen into one phage. In addition, a phage will be engineered that contains mixed tail fibers specific to Salmonella and E. coli to demonstrate the engineering of the phages' host range. The proposed research is significant because while phages have evolved to be near perfect predators of specific bacteria, practical hurdles have limited their use for pathogen detection and treatment. By mitigating these hurdles, significant advances toward human health and safety can be achieved using genetically engineered phages.